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Keywords:

  • ATPase;
  • cation diffusion facilitator (CDF) transporter family;
  • cation/ H+ antiport;
  • manganese;
  • membrane transport;
  • metals;
  • Nramp transporter family;
  • ZRT/IRT1-related protein (ZIP) transporter family

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Mechanisms of Mn2+ accumulation into the cell
  5. Whole-plant Mn2+ uptake characteristics compared with emerging insights from the molecular data
  6. Mechanisms of Mn2+ accumulation into endomembrane compartments
  7. Mechanisms of Mn2+ efflux from the cell
  8. Regulation of Mn2+ transport
  9. Identification of molecular determinants of Mn2+ specificity
  10. Mn homeostasis mutants and hyperaccumulators
  11. Conclusions and future perspectives
  12. Acknowledgements
  13. References

Manganese (Mn) is an essential metal nutrient for plants. Recently, some of the genes responsible for transition metal transport in plants have been identified; however, only relatively recently have Mn2+ transport pathways begun to be identified at the molecular level. These include transporters responsible for Mn accumulation into the cell and release from various organelles, and for active sequestration into endomembrane compartments, particularly the vacuole and the endoplasmic reticulum. Several transporter gene families have been implicated in Mn2+ transport, including cation/H+ antiporters, natural resistance-associated macrophage protein (Nramp) transporters, zinc-regulated transporter/iron-regulated transporter (ZRT/IRT1)-related protein (ZIP) transporters, the cation diffusion facilitator (CDF) transporter family, and P-type ATPases. The identification of mutants with altered Mn phenotypes can allow the identification of novel components in Mn homeostasis. In addition, the characterization of Mn hyperaccumulator plants can increase our understanding of how plants can adapt to excess Mn, and ultimately allow the identification of genes that confer this stress tolerance. The identification of genes responsible for Mn2+ transport has substantially improved our understanding of plant Mn homeostasis.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Mechanisms of Mn2+ accumulation into the cell
  5. Whole-plant Mn2+ uptake characteristics compared with emerging insights from the molecular data
  6. Mechanisms of Mn2+ accumulation into endomembrane compartments
  7. Mechanisms of Mn2+ efflux from the cell
  8. Regulation of Mn2+ transport
  9. Identification of molecular determinants of Mn2+ specificity
  10. Mn homeostasis mutants and hyperaccumulators
  11. Conclusions and future perspectives
  12. Acknowledgements
  13. References

Manganese (Mn) is an essential nutrient in most, if not all, organisms. It is of particular importance in photosynthetic organisms where a cluster of Mn atoms is required as the catalytic centre for light-induced water oxidation in photosystem II, and is required as a cofactor for a variety of enzymes, such as the Mn2+-dependent superoxide dismutase (MnSOD) (Marschner, 1995). Despite its importance, the amount of Mn that is required by a plant is relatively low, yet the capacity for Mn uptake greatly exceeds this requirement (Clarkson, 1988). Mn can be particularly toxic to plant growth and a variety of mechanisms exist to overcome such toxicity, including the conversion of the metal to a metabolically inactive compound, such as a Mn2+-chelate complex, or sequestration of the Mn2+ ion or a Mn2+-chelate complex into an internal compartment such as the vacuole. At the cellular level, Mn2+ accumulates predominantly in the vacuole and to some extent in chloroplasts, and can be associated with the cell wall fraction (McCain & Markley, 1989; Quiquampoix et al., 1993; González & Lynch, 1999). For instance, a high concentration (10 µm) of free Mn2+ was measured in vacuoles following exposure of maize (Zea mays) roots to high concentrations (10–100 µm) of external Mn (Quiquampoix et al., 1993). In another study, a large accumulation of Mn2+ (∼80% of total Mn) was observed in vacuoles from leaves of Phaseolus vulgaris grown in excess Mn (200 µm) conditions, in addition to a significant accumulation of Mn2+ in chloroplasts, particularly in young leaves (González & Lynch, 1999). In both studies, the external Mn concentrations used were equivalent to those found in Mn-toxic acid soils. The distribution of Mn2+ amongst these fractions, particularly between vacuolar and chloroplast fractions, can vary depending on tissue type and the developmental stage of the tissue, as well as the degree of Mn stress to which the plant is exposed. Mn is also required in other organelles. MnSOD has been identified in peroxisomes and mitochondria, and a variety of Golgi-localized enzymes, such as glycosyltransferases, require Mn2+ (Marschner, 1995). Additionally, Mn2+ has been shown to accumulate into the endoplasmic reticulum (Wu et al., 2002). Thus, Mn2+ will be exported to other endomembrane locations in addition to the chloroplast and vacuole, although its accumulation into other organelles is minor compared to Mn2+ concentrations in the vacuole.

The mechanisms of Mn2+ transport and homeostasis are well understood in microorganisms; in many bacterial species, and in the yeast Saccharomyces cerevisiae, several Mn2+ transport pathways have been identified at the molecular level (Jakubovics & Jenkinson, 2001; Luk et al., 2003b; Fig. 1a). It has been observed that many of these transporters for Mn2+ have broad substrate specificities. This may be a result in part of the equivalent characteristics of some cations. The ionic radius of Mn2+ is between that of Ca2+ and Mg2+, and close to that of Fe2+. There is also coordination between Mn and iron (Fe) homeostasis, which is particularly evident in some bacterial systems. Fe can contribute to the production of reactive oxygen species by reacting with H2O2 to form hydroxyl radicals, while Mn contributes to the detoxification of reactive oxygen species as a cofactor for MnSOD and Mn-dependent catalases, as well as by the ability of simple Mn(II) salts to catalyse the dismutation of superoxide radicals (Jakubovics & Jenkinson, 2001). This importance of Mn in oxidative stress resistance is particularly apparent in bacteria such as Staphylococcus aureus and Bacillus subtilis that only possess the MnSOD enzyme and have no FeSOD activity, unlike in yeast and plants that have SOD activities dependent on various metals. Thus it has been proposed that such bacterial cells have to ensure an accurate balance between these two metals. Many bacteria have sophisticated mechanisms for coordination of Fe sensing, Mn sensing and oxidative stress response. For example, in S. aureus and B. subtilis a coordinated network of metalloproteins, including an Fe2+ uptake regulator (Fur), a Mn2+ transport regulator (MntR) and an Fe2+/Mn2+-dependent peroxide regulator (PerR), sense and regulate intracellular Fe2+ and Mn2+ concentrations, sense H2O2 concentrations, and finally regulate, via PerR, oxidative stress response genes (Herbig & Helmann, 2001; Horsburgh et al., 2002).

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Figure 1. A diagram of a typical cell comparing Mn2+ transport pathways in the yeast Saccharomyces cerevisiae (a) and the plant Arabidopsis thaliana (b). (a) The currently identified yeast Mn2+ transport pathways are: SMF1, a plasma membrane Nramp Mn2+ transporter; PHO84, a plasma membrane phosphate transporter that can transport Mn2+ under excess Mn stress conditions; PMR1, a Golgi Ca2+- and Mn2+-transporting P-type ATPase; SMF2, an intracellular vesicle Nramp Mn2+ transporter; CCC1, a Mn2+ and Fe2+ vacuolar transporter; and MTM1, the mitochondrial inner membrane protein member of the mitochondrial carrier family, which delivers Mn2+ to the mitochondrial superoxide dismutase SOD2. These transport pathways are reviewed further in Luk et al. (2003b). (b) The currently identified Arabidopsis Mn2+ transport pathways are: AtIRT1, a plasma membrane ZRT/IRT1-related protein (ZIP) family transporter that can transport Mn2+ plus Fe2+, Zn2+ and Cd2+ under Fe-deficiency conditions; AtECA1, an endoplasmic reticulum (ER) Ca2+- and Mn2+-transporting P-type ATPase; the cation exchanger (CAX) AtCAX2, a vacuolar cation/H+ antiporter that can transport Mn2+ plus Ca2+ and Cd2+ AtNramp3, a vacuolar Nramp transporter that can transport Mn2+ plus Fe2+ and Cd2+ in Fe-deficiency conditions. Arabidopsis may possess a vacuolar-localized cation diffusion facilitator (CDF) family transporter related to the Stylosanthes hamata Mn2+ transporter ShMTP1. Mechanisms for Mn2+ transport into Golgi, mitochondria and chloroplast, in addition to Mn2+ efflux out of the cell, are currently unknown. It is yet to be confirmed whether Arabidopsis has a plasma membrane-localized Nramp transporter analogous to SMF1. The oligopeptide transporter-like protein AtOPT3 may be involved in Mn2+ accumulation, in addition to the possible transport of Cu2+ and Fe2+. Arabidopsis may have a yellow stripe-like (AtYSL) transporter with equivalent function to rice OsYSL2, a Mn2+-nicotianamine (NA) and Fe2+-NA transporter.

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Various physiological and biochemical studies performed in the 1960s to 1980s provided some details as to how Mn2+ is accumulated and transported in higher plants (reviewed by Clarkson, 1988; Rengel, 2000). However, since those experiments, plant Mn2+ transport mechanisms have received little attention. In the last few years there have been large advances in the molecular characterization of transition metal transporters and many metal transport pathways have now been identified at the molecular level in plants, particularly for Fe2+, Zn2+ and Cu2+ transport (Hall & Williams, 2003). Only relatively recently have we begun to determine the wide range of transporters that are involved in higher plant Mn2+ transport (Fig. 1b). In this article, these emerging Mn2+ transport pathways are reviewed. Whole-plant, long-distance translocation of Mn has been reviewed elsewhere (Loneragan, 1988; Rengel, 2000), and so this review will put particular emphasis on the mechanisms of Mn2+ transport at the cellular level. I shall describe the proteins that mediate Mn2+ transport into the cell, and those that transport Mn2+ into various organelles, and contrast these with the well-described Mn2+ transport mechanisms in microorganisms, particularly the yeast S. cerevisiae. I shall also discuss how the analysis of plants with altered Mn homeostasis can be an additional means to identify genes involved in Mn2+ transport and homeostasis. Finally, I shall assess the current understanding of plant Mn nutrition and discuss future directions.

Mechanisms of Mn2+ accumulation into the cell

  1. Top of page
  2. Summary
  3. Introduction
  4. Mechanisms of Mn2+ accumulation into the cell
  5. Whole-plant Mn2+ uptake characteristics compared with emerging insights from the molecular data
  6. Mechanisms of Mn2+ accumulation into endomembrane compartments
  7. Mechanisms of Mn2+ efflux from the cell
  8. Regulation of Mn2+ transport
  9. Identification of molecular determinants of Mn2+ specificity
  10. Mn homeostasis mutants and hyperaccumulators
  11. Conclusions and future perspectives
  12. Acknowledgements
  13. References

In common with other transition metals, such as Fe and copper (Cu), Mn can exist in various oxidation states. The availability of Mn to the plant depends on its oxidation state, as the oxidized forms Mn(III) and Mn(IV) are not bioavailable to plants and cannot be accumulated (Clarkson, 1988; Rengel, 2000). It is the reduced form of Mn(II) that is transported (as the divalent cation Mn2+) into the cell. There is also evidence that Mn can be taken up into the root cell as a Mn2+-ethylenediaminetetraacetic acid (EDTA) complex in some species (Laurie et al., 1995). The concentration of Mn2+ in the soil solution can vary markedly, depending on the soil solution pH. Acidification of the rhizosphere by exudation of H+ or organic acids increases the availability of Mn2+ for accumulation into the cell. However, when soils are too acidic (< pH 5.5), Mn2+ toxicity can be a critical limiting factor to plant growth; similarly, Mn2+ deficiency can be a problem with alkaline soils (Marschner, 1995). It has been suggested that the plasma membrane ferric chelate reductase can reduce Mn(III) under Fe-deficiency conditions, which stimulate the reductase activity, although ferric chelate reductase activity itself is not stimulated by Mn deficiency (Norvell et al., 1993; Delhaize, 1996). Both Fe and zinc (Zn) deficiency can enhance Mn2+ uptake in some species (Welch & Norvell, 1993; Cohen et al., 1998). In contrast, excess Fe and cadmium (Cd) have been shown to inhibit Mn2+ accumulation (Roomizadeh & Karimian, 1996; Hernandez et al., 1998), indicating that, in plants, broad-specificity transporters may be responsible for Mn2+ accumulation.

Our understanding of plant Mn2+ transport mechanisms may be greatly aided by inferring knowledge from the yeast S. cerevisiae. High-affinity Mn2+ accumulation into the yeast cell occurs principally via the natural resistance-associated macrophage protein (Nramp) family transporter suppressor of mif1 (SMF1) (Supek et al., 1996; Fig. 1a). Nramp transporters appear to be evolutionarily conserved throughout organisms, including plants, and to date Nramp genes have been cloned and characterized from various plants including rice (Oryza sativa), soybean (Glycine max), tomato (Lycopersicon esculentum) and Arabidopsis thaliana (Thomine et al., 2000; Bereczky et al., 2003; Kaiser et al., 2003). By analogy to yeast, it may be expected that some plant Nramps function in Mn2+ accumulation. AtNramp1, AtNramp3 and AtNramp4 can all complement a yeast mutant lacking SMF1, indicating that all three proteins can transport Mn2+ (Thomine et al., 2000). Similarly, LeNramp1 and LeNramp3 can also complement the smf1 strain (Bereczky et al., 2003). However, as in other organisms, plant Nramps are not specific to a single metal substrate; for example, AtNramp3 can also transport Fe2+, and probably Zn2+ and Cd2+ (Thomine et al., 2000; Thomine et al., 2003), and a soybean Nramp, GmDMT1 (divalent metal transporter 1), can transport Fe2+ in addition to Mn2+, and possibly Zn2+ and Cu2+ (Kaiser et al., 2003). It should be noted, however, that, apart from Fe2+ transport by AtNramp3 and GmDMT1, direct transport measurements have not been reported for plant Nramps, particularly with respect to Mn2+ transport. The AtNramp and LeNramp genes are up-regulated by Fe deficiency, indicating an in vivo role for the plant Nramp transporters in Fe homeostasis. However, in addition to Fe homeostasis, a possible role for AtNramp3 in Mn homeostasis has been indicated. Knockout of AtNramp3 induced a significant increase in Mn content in seedlings under Fe deficiency, while AtNramp3 overexpression decreased Mn content (Thomine et al., 2003). These phenotypes were explained by the internal localization of AtNramp3, which was shown to be localized at the tonoplast rather than the plasma membrane, and suggested a role in vacuolar metal release (Thomine et al., 2003). To date, no plant Nramp has been found to be plasma membrane localized, and therefore an Nramp transporter for catalysing cellular Mn2+ uptake as seen with SMF1 has yet to be found, highlighting the importance of determining the membrane localization of the plant Nramps.

An alternate mechanism of Mn2+ accumulation into S. cerevisiae is via the high-affinity phosphate transporter PHO84 (Fig. 1a). It has been suggested that PHO84 functions as a low-affinity Mn transporter, possibly in transporting MnHPO4, when the yeast is exposed to high toxic concentrations of external Mn (Luk et al., 2003b). Plants such as Arabidopsis possess various phosphate transporters with significant similarity to PHO84, such as phosphate transporter 1 (AtPT1) and AtPT2. There is currently no evidence for Mn2+ or MnHPO4 accumulation by a plant phosphate transporter, although this remains an intriguing possibility.

There are various additional pathways by which Mn2+ could potentially accumulate into the plant cell. It has been postulated that plasma membrane Ca2+ channels may allow the uptake of Mn2+, and indeed some channels are permeable to a range of cations including Mn2+ (White, 1998). In many organisms, members of the ZIP [zinc-regulated transporter/iron-regulated transporter (ZRT/IRT1)-related protein] transporter family are involved in uptake of metals into the cell. For example, the ZIP transporter ZRT1 mediates accumulation of Zn2+ into the yeast cell and an Arabidopsis ZIP transporter, IRT1, mediates the uptake of Fe2+ into the root cell (Mäser et al., 2001). Many of the plant ZIP transporters have been found to have broad substrate specificities. For example, Arabidopsis IRT1 can transport a range of metals as well as Fe2+, including Cd2+, Zn2+ and Mn2+ (Korshunova et al., 1999). A knockout mutant of IRT1 clearly demonstrates that this transporter is the critical pathway for Fe acquisition during Fe deprivation into the Arabidopsis root (Vert et al., 2002). The severe morphological phenotype of irt1 is rescued by application of exogenous Fe. Although application of Mn does not rescue the phenotype, the irt1 mutant plant has a complete reduction in root Mn concentrations compared to wild-type when grown under Fe-deficiency conditions, indicating that IRT1 can function as a Mn2+ uptake transporter in planta, and that it is the primary pathway for Mn2+ transport during Fe deficiency. IRT1 is only up-regulated upon Fe deficiency; therefore the potential for Mn2+ accumulation via IRT1 will be limited to particular environmental conditions. Interestingly, current analysis suggests that IRT1 is the only member of the 15 Arabidopsis ZIP transporters that can transport Mn2+ (Mäser et al., 2001), although transport characteristics for many of the other Arabidopsis ZIPs have yet to be reported. In contrast, multiple members of ZIP transporter families from other plant species appear to have the ability to transport Mn2+, such as MtZIP4 and MtZIP7 from Medicago truncatula (López-Millán et al., 2004) and LeIRT1 and LeIRT2 from tomato (Eckhardt et al., 2001).

Maize yellow stripe (YS) and yellow stripe-like (YSL) proteins are involved in metal-complex transport at the plasma membrane in a range of plants including maize, rice and Arabidopsis (Roberts et al., 2004). Many grass species use phytosiderophores (PSs) to chelate metals in the soil environment before accumulation into the roots, and it is a metal–PS complex that is transported by ZmYS1. Nongrass species including Arabidopsis do not produce PS but do utilize nicotianamine (NA), which is a potent chelator of various transition metals, particularly Fe2+ but also including Mn2+. Metal–NA complexes are therefore potential substrates for YSL transporters. Although Fe2+ is the predominant metal transported for all the YSL proteins characterized to date, there is some evidence that Mn2+ complexes may be transported by some proteins, including OsYSL2 from rice (Koike et al., 2004) and to some extent ZmYS1 from maize (Schaaf et al., 2004), although other data also indicate that Mn2+ is not a substrate for ZmYS1 (Roberts et al., 2004). OsYSL2 can transport Mn2+-NA and Fe2+-NA to equal extents, as determined in an oocyte expression transport activity assay. In addition, localization analysis demonstrated that OsYSL2 transports Fe2+-NA and Mn2+-NA across the plasma membrane for internalization and for distribution into the phloem for long-distance transport (Koike et al., 2004). There are eight predicted YSL proteins in Arabidopsis, and while many remain to be characterized in detail, there is as yet no evidence for Mn2+-complex transport by any of these proteins. A putative oligopeptide transporter (OPT) AtOPT3 shares sequence similarity with the ZmYS1 and AtYSL genes. AtOPT3 transcript is up-regulated dramatically in root tissue by Mn-deficiency conditions, and AtOPT3 can restore growth of the smf1 Mn-sensitive yeast strain (Wintz et al., 2003). This indicates a role of AtOPT3 in cation transport, although it has yet to be determined if this putative transporter facilitates direct transport of a metal or metal complex. Expression of AtOPT3 in vascular tissue suggests a role in long-distance transport of metals, and AtOPT3 also appears to be essential for embryo development (Stacey et al., 2002).

Whole-plant Mn2+ uptake characteristics compared with emerging insights from the molecular data

  1. Top of page
  2. Summary
  3. Introduction
  4. Mechanisms of Mn2+ accumulation into the cell
  5. Whole-plant Mn2+ uptake characteristics compared with emerging insights from the molecular data
  6. Mechanisms of Mn2+ accumulation into endomembrane compartments
  7. Mechanisms of Mn2+ efflux from the cell
  8. Regulation of Mn2+ transport
  9. Identification of molecular determinants of Mn2+ specificity
  10. Mn homeostasis mutants and hyperaccumulators
  11. Conclusions and future perspectives
  12. Acknowledgements
  13. References

As discussed earlier, there is still a scarcity of physiological and biochemical data describing the uptake kinetics of Mn2+ by plant roots. From the studies that have been performed, estimates of the Km for low-affinity Mn2+ uptake into roots range between 4 and 400 µm (reviewed in Rengel, 2000). Some of this variation is probably attributable to the different plant systems used, but is also attributable to the different experimental conditions with regard to the external Mn concentrations used. Many studies used nutrient solutions with Mn supplied at typically 1–5 µm, although some earlier uptake studies did use unrealistically high Mn concentrations of 1–10 mm, compared to concentrations of 0.1–800 µm Mn normally found in the soil solution, and therefore only examined low-affinity uptake pathways (Clarkson, 1988; Rengel, 2000). It is interesting to compare these whole-plant data with the molecular information we now have for plant Mn2+ accumulation. Kinetic studies with heterologously expressed IRT1 in yeast demonstrate that IRT1-dependent Mn2+ uptake is concentration-dependent and saturable, with an apparent Km of 9 ± 1 µm (Korshunova et al., 1999), which is consistent with Km measurements obtained from whole-plant studies using realistic external Mn concentrations (Landi & Fagioli, 1983). The accumulation of Mn2+ into root cells is driven, at least in part, by the electrochemical potential difference across the plasma membrane, as observed in whole-plant studies in which the addition of an uncoupler or NaN3 strongly inhibited Mn2+ accumulation (Clarkson, 1988). It would therefore be expected that some cation influx transporters are H+ coupled. Indeed, ZmYS1 is energized by H+-cotransport (Schaaf et al., 2004), which may also be true for other related YSL/OPT-type transporters. Similarly, it has been demonstrated that Nramp transporters are H+-coupled (Hall & Williams, 2003). A number of physiological studies have observed that other metals, including Fe and Cd, can significantly inhibit the accumulation of Mn2+ into plants (Roomizadeh & Karimian, 1996; Hernandez et al., 1998). This correlates well with the observation from the molecular data that many Mn2+ influx transporters have broad selectivity.

Mechanisms of Mn2+ accumulation into endomembrane compartments

  1. Top of page
  2. Summary
  3. Introduction
  4. Mechanisms of Mn2+ accumulation into the cell
  5. Whole-plant Mn2+ uptake characteristics compared with emerging insights from the molecular data
  6. Mechanisms of Mn2+ accumulation into endomembrane compartments
  7. Mechanisms of Mn2+ efflux from the cell
  8. Regulation of Mn2+ transport
  9. Identification of molecular determinants of Mn2+ specificity
  10. Mn homeostasis mutants and hyperaccumulators
  11. Conclusions and future perspectives
  12. Acknowledgements
  13. References

The sequestration of metals into endomembrane compartments is a critical means of providing tolerance to elevated and toxic concentrations of various metals, including Mn. The vacuole is a dynamic organelle that comprises as much as 90% of the total cell volume in some cell types, and is therefore the predominant sink for many toxic compounds. Kinetic analysis and nuclear magnetic resonance (NMR) spectroscopy on whole-plant tissue has demonstrated that the vacuole is the major internal Mn store (Clarkson, 1988). Of the Mn2+ transport pathways that have been elucidated at the molecular level, it is those mediating uptake into this organelle that have been the best characterized. Arabidopsis has a multigene family of cation exchanger (CAX) genes, which includes the tonoplast-localized cation/H+ antiporter CAX2 (Mäser et al., 2001). CAX transporters, including CAX2, were originally identified as Ca2+ transporters; however, CAX2 also has the ability to transport Mn2+, as shown by expression studies in tobacco (Nicotiana tabacum) and yeast (Hirschi et al., 2000; Schaaf et al., 2002; Shigaki et al., 2003). When ectopically expressed in tobacco, CAX2 can mediate tolerance to otherwise toxic concentrations of Mn (Hirschi et al., 2000). Similarly, heterologous expression of CAX2 in yeast can induce Mn tolerance (Schaaf et al., 2002; Shigaki et al., 2003). Indeed, in a yeast Mn hypersensitivity suppression screen, CAX2 was the only gene identified out of over 100 000 transformants that could suppress the Mn toxicity phenotype (Schaaf et al., 2002). This tolerance is a result of direct transport of Mn2+ by CAX2 (Shigaki et al., 2003). Recently, CAX2 has been confirmed to have a critical role in vacuolar Mn2+ transport in planta. Analysis of CAX2 T-DNA knockout lines found a significant decrease in Mn2+ transport into vacuolar membrane vesicles compared to wild-type plants, while Ca2+ transport was not significantly affected (Pittman et al., 2004b). Furthermore, analysis of CAX2 promoter–β-glucuronidase (GUS) reporter gene fusions has indicated strong expression of CAX2 in vascular tissue throughout the plant (Pittman et al., 2004b), particularly the phloem (J. K. Pittman, unpublished), indicating a possible role of CAX2 in regulating loading or unloading of Mn from the vasculature. An orthologue of CAX2 from rice can transport Mn2+ (Kamiya & Maeshima, 2004) and Mn2+/H+ antiport activity has been determined in Avena sativa vacuolar membrane preparations (González et al., 1999), indicating that in many plant species vacuolar sequestration of Mn2+ is mediated by CAX-like transporters. CAX transporters are low-affinity cation transporters; for example, CAX2 has a low affinity for Mn2+ (Shigaki et al., 2003). This suggests that the role of CAX-like transporters in Mn2+ transport may be restricted to conditions of Mn stress and toxicity when cytosolic Mn2+ concentrations are significantly elevated, as demonstrated by the ability of CAX2 to provide tolerance to tobacco plants grown under conditions of considerable Mn toxicity (Hirschi et al., 2000).

CAX transporters are not unique in mediating Mn2+ transport into the plant vacuole. In the Mn-tolerant tropical legume Stylosanthes hamata, vacuolar Mn2+ transport is mediated by ShMTP1 (metal tolerance protein), a member of the cation diffusion facilitator (CDF) transporter family (also referred to as the cation efflux family) (Delhaize et al., 2003). ShMTP1 was able to promote Mn tolerance, but not tolerance to any other metal, when heterologously expressed both in yeast and in Arabidopsis, and ShMTP1-green fluorescent protein (GFP) was shown to localize to the tonoplast in tobacco and Arabidopsis plants. As with other CDF family members, ShMTP1 is proposed to transport its substrate by H+ cotransport, although Mn2+/H+ transport has yet to be directly demonstrated. In addition, three other related transporters, ShMTP2, ShMTP3 and ShMTP4, were able to confer tolerance to high concentrations of Mn in yeast (Delhaize et al., 2003). As yet, no CDF transporters from a plant other than S. hamata have been shown to transport Mn2+, although putative transporters with high sequence similarity to ShMTP1 are found in Arabidopsis and rice.

In S. cerevisiae, a transporter named Ca2+-sensitive cross-complementer 1 (CCC1) has been implicated in Mn2+ sequestration into the vacuole (Lapinskas et al., 1996; Li et al., 2001; Fig. 1a). Originally, localization of CCC1 was proposed to be to the Golgi, but it was subsequently shown to be a vacuolar membrane protein, and thought to function predominantly as a Fe2+ transporter (Li et al., 2001). Interestingly, plants do possess homologues to CCC1; for example, Arabidopsis has a single gene closely related to yeast CCC1. Another S. cerevisiae protein called manganese trafficking factor for mitochondrial SOD2 (MTM1), which is a member of the mitochondrial carrier family (MCF) of proteins, is present at the inner mitochondrial membrane (Fig. 1a) and is required for delivering Mn2+ to the mitochondrial matrix-localized Mn2+-dependent SOD2 (Luk et al., 2003a). It was concluded that MTM1 is not responsible for a general accumulation of Mn2+ into the mitochondrial matrix, but is specifically required for trafficking Mn2+ to SOD2, possibly by functioning as a SOD2-specific Mn2+ chaperone. As in yeast and humans, MCF proteins are present in plants; Arabidopsis has nearly 60 putative MCF members, although the functions of most are still unknown (Picault et al., 2004).

Another major pathway for intracellular Mn2+ sequestration in yeast is transport into the Golgi by the Ca2+- and Mn2+-transporting ATPase plasma membrane ATPase related 1 (PMR1) (Dürr et al., 1998). It is still unclear by which mechanism Mn2+ is transported into the plant Golgi; however, an Arabidopsis endoplasmic reticulum-localized Ca2+-ATPase, ECA1, with homology to PMR1, can also transport Mn2+. A T-DNA knockout of ECA1, grown on high-Mn media, displays a strong stress phenotype when compared to wild-type plants (Wu et al., 2002). This phenotype includes a significant reduction in fresh weight, dramatic leaf chlorosis, a significant inhibition of leaf expansion and root elongation, and a loss of root hair tip growth. The role of ECA1 in Mn2+ transport was further confirmed by its ability to confer tolerance to toxic concentrations of Mn when heterologously expressed in a Mn-sensitive mutant yeast strain (Wu et al., 2002). Although direct Mn2+ transport by ECA1 awaits confirmation, this study is a major advance in our understanding of endomembrane Mn homeostasis.

Mn has a critical role in the water oxidation step of photosynthesis, and the chloroplast is the second-largest sink for Mn in the cell. However, the mechanisms of Mn2+ accumulation by the chloroplast are unknown. Some insight into Mn2+ accumulation in higher plant chloroplasts may be inferred from the well-understood Mn2+ transport mechanisms in photosynthetic cyanobacteria such as Synechocystis sp. PCC 6803. Cyanobacteria accumulate Mn2+ via a high-affinity Mn2+ transporter of the ATP-binding cassette (ABC) transporter superfamily (MntABC), which transports Mn2+ across the inner membrane and is induced under Mn-starved conditions (Bartsevich & Pakrasi, 1996). Synechocystis 6803 also has additional Mn2+ accumulation mechanisms for which the genes have not been identified. These include a membrane potential-dependent and light-dependent process that mediates a Mn2+ transport step across the outer membrane into the periplasm, and then a second light-independent transport step across the inner membrane (Keren et al., 2002).

Mechanisms of Mn2+ efflux from the cell

  1. Top of page
  2. Summary
  3. Introduction
  4. Mechanisms of Mn2+ accumulation into the cell
  5. Whole-plant Mn2+ uptake characteristics compared with emerging insights from the molecular data
  6. Mechanisms of Mn2+ accumulation into endomembrane compartments
  7. Mechanisms of Mn2+ efflux from the cell
  8. Regulation of Mn2+ transport
  9. Identification of molecular determinants of Mn2+ specificity
  10. Mn homeostasis mutants and hyperaccumulators
  11. Conclusions and future perspectives
  12. Acknowledgements
  13. References

It has been suggested that Mn2+ may be exported from the cell via a Mn2+/H+ antiport mechanism (Clarkson, 1988), although as yet there is no biochemical or genetic evidence for this. ATP-dependent transporters such as members of the P-type ATPase and ABC transporter families mediate active efflux of substrates across the plasma membrane. A subfamily of P-type ATPases, the P1B-ATPases, catalyse transition metal efflux in many organisms including plants, and are predicted to transport either Zn2+/Cd2+/Pb2+/Co2+ or Cu2+/Ag2+ (Hall & Williams, 2003), but there is no evidence that Mn2+ is a substrate for P1B-ATPases from any organism. Arabidopsis ECA1 and S. cerevisiae PMR1 can transport Mn2+ in addition to Ca2+ (see previous section), so it is conceivable that other members of this P2A-ATPase subgroup have similar substrate specificity. ECA1 is located at the endoplasmic reticulum, but the membrane localization of the three Arabidopsis homologues, ECA2 to ECA4, has yet to be determined. ABC transporters are responsible for a wide range of cellular detoxification processes, such as providing tolerance to secondary metabolites and heavy metals (Martinoia et al., 2002; Hall & Williams, 2003). Mn2+-transporting ABC transporters are present in many bacteria (Jakubovics & Jenkinson, 2001), but there is as yet no definitive evidence for any cation transport by plant ABC transporters. However, plant genomes such as Arabidopsis and rice have a very large number of ABC transporters, of which many remain to be characterized. There is some biochemical evidence to suggest the presence of ATP-dependent and vanadate-sensitive Mn2+ transport in Arabidopsis. The Arabidopsis IAA-leucine resistant 2 (ilr2) mutant has a slight tolerance to Mn stress. Transport characterization of microsomal membrane vesicles from ilr2 plants demonstrated a significant increase in ATP-dependent Mn2+ transport compared to wild-type plants (Magidin et al., 2003). The ILR2 gene was cloned and encodes a novel soluble protein, and so is not a transporter itself. It was proposed that ILR2 might act as a regulator of Mn2+ transport, possibly acting on Mn2+ efflux from the cell mediated by either an ATPase or an ABC transporter (Magidin et al., 2003).

Regulation of Mn2+ transport

  1. Top of page
  2. Summary
  3. Introduction
  4. Mechanisms of Mn2+ accumulation into the cell
  5. Whole-plant Mn2+ uptake characteristics compared with emerging insights from the molecular data
  6. Mechanisms of Mn2+ accumulation into endomembrane compartments
  7. Mechanisms of Mn2+ efflux from the cell
  8. Regulation of Mn2+ transport
  9. Identification of molecular determinants of Mn2+ specificity
  10. Mn homeostasis mutants and hyperaccumulators
  11. Conclusions and future perspectives
  12. Acknowledgements
  13. References

In bacteria, the accumulation of transition metals including Mn2+ is tightly regulated by well-understood transcriptional regulation mechanisms (Jakubovics & Jenkinson, 2001). Similarly, in yeast there are mechanisms for the transcriptional regulation of Fe2+, Zn2+ and Cu2+ transport and homeostasis; however, transcriptional regulation of Mn2+ accumulation has not as yet been identified (Van Ho et al., 2002). S. cerevisiae does have an apparently novel mechanism of post-translational regulation of Mn2+ transport by SMF1 and SMF2 (Fig. 1a). Under Mn-abundant conditions, SMF1 and SMF2 are targeted to the vacuole for degradation by bypass SOD deficiency 2 (BSD2), an endoplasmic reticulum-localized protein (Van Ho et al., 2002). Under Mn-starved conditions, SMF1 is not recognized by BSD2 and is targeted to the plasma membrane to facilitate Mn2+ accumulation. Clarkson (1988) noted that many plant species have particularly high uptake rates compared with the growth requirement for Mn, indicating that Mn2+ accumulation is not as tightly regulated in plants as in microorganisms.

Despite the identification of various candidate genes responsible for Mn2+ transport, it is still largely unclear how plant Mn2+ transport is regulated. As described in previous sections, many of the broad-specificity transporters that can also transport Mn2+, such as IRT1 and AtNramp3, are up-regulated under Fe-deficiency conditions. These transporters do not appear to be regulated by external Mn concentrations, but they both regulate the Fe-starvation-dependent accumulation of Mn2+ into the plant (Vert et al., 2002; Thomine et al., 2003). AtOPT3 is the first example of a putative metal transporter that is strongly up-regulated by Mn deficiency, although this regulation is not specific for Mn as AtOPT3 is also up-regulated to some extent by Fe and Cu deficiency (Wintz et al., 2003). As there is a high capacity for Mn2+ accumulation into the cell, it is therefore likely that mechanisms for Mn2+ sensing and transport from the cytosol, either outward efflux or internal sequestration, are tightly controlled. Further work is required to determine whether Mn2+ transport activity by ECA1 and ShMTP1 is a regulated process, and if so by what mechanism. Transport of Mn2+ into the vacuole by CAX2 may be regulated by a mechanism involving an N-terminal region of the protein. When heterologously expressed in yeast, CAX2 is unable to mediate Mn2+ transport unless the protein is N-terminally truncated (Schaaf et al., 2002; Pittman et al., 2004b). It is suggested that autoinhibition by the N-terminus of CAX2 disrupts transport activity; however, this is not specific for Mn2+ as transport of Ca2+ by CAX2 is similarly affected (Pittman et al., 2004b).

Identification of molecular determinants of Mn2+ specificity

  1. Top of page
  2. Summary
  3. Introduction
  4. Mechanisms of Mn2+ accumulation into the cell
  5. Whole-plant Mn2+ uptake characteristics compared with emerging insights from the molecular data
  6. Mechanisms of Mn2+ accumulation into endomembrane compartments
  7. Mechanisms of Mn2+ efflux from the cell
  8. Regulation of Mn2+ transport
  9. Identification of molecular determinants of Mn2+ specificity
  10. Mn homeostasis mutants and hyperaccumulators
  11. Conclusions and future perspectives
  12. Acknowledgements
  13. References

Many metal transporters appear to have broad substrate ranges, but little is known regarding the molecular mechanisms that determine substrate specificity or how transporters can be engineered to alter this specificity. Some of the molecular determinants of the Mn2+ specificity of transport proteins have been determined. Recent studies have demonstrated that single or few amino acid changes can alter the metal specificity of metal transporters. Specific amino acid changes to the Arabidopsis and rice Ca2+–Mn2+/H+ antiporters CAX2 and OsCAX1a can completely abolish Mn2+ transport while retaining Ca2+ transport (Shigaki et al., 2003; Kamiya & Maeshima, 2004). In contrast, a single amino acid substitution on the yeast CAX-type transporter vacuolar H+/Ca2+ exchanger (VCX1), a vacuolar Ca2+/H+ antiporter, can specifically introduce Mn2+ transport activity while retaining Ca2+ transport (Pittman et al., 2004a). Similarly, specific amino acids have been identified that determine the Mn2+ specificity of the yeast Ca2+–Mn2+ ATPase PMR1, of Arabidopsis IRT1, and of S. hamata ShMTP1 (Mandal et al., 2000; Rogers et al., 2000; Delhaize et al., 2003). When Arg-123 of ShMTP1 is mutated to Ile, the ability to confer Mn tolerance to either yeast or Arabidopsis is completely lost (Delhaize et al., 2003). Substitution of Asp-100 or Asp-136 with Ala in IRT1 eliminates the ability of IRT1 to complement both Fe- and Mn-sensitive yeast mutants, but retains the ability to complement a Zn-sensitive yeast strain (Rogers et al., 2000). Substitution of Gln-783 with Ala within a transmembrane-spanning domain of PMR1 causes a selective loss of Mn2+ transport activity (Mandal et al., 2000). The ability to manipulate metal transporters, such as by altering substrate specificity, is an essential step in developing genetically engineered plants that can be used for phytoremediation strategies for specific metals.

Mn homeostasis mutants and hyperaccumulators

  1. Top of page
  2. Summary
  3. Introduction
  4. Mechanisms of Mn2+ accumulation into the cell
  5. Whole-plant Mn2+ uptake characteristics compared with emerging insights from the molecular data
  6. Mechanisms of Mn2+ accumulation into endomembrane compartments
  7. Mechanisms of Mn2+ efflux from the cell
  8. Regulation of Mn2+ transport
  9. Identification of molecular determinants of Mn2+ specificity
  10. Mn homeostasis mutants and hyperaccumulators
  11. Conclusions and future perspectives
  12. Acknowledgements
  13. References

Screening and characterization of mutants with altered Mn phenotypes can allow the identification of novel components in Mn homeostasis. The Arabidopsis manganese accumulator 1 (man1) mutant was originally identified as a Mn hyperaccumulator (Delhaize, 1996). man1 has a 7.5-fold increase in leaf Mn content compared to wild-type and a 10-fold increase in root Mn content. In addition to Mn, man1 also hyperaccumulates other metals, including Fe. For example, there was a 12-fold increase in Fe content in the roots of man1, which correlates with high, constitutive ferric-chelate reductase activity (Delhaize, 1996; Rogers & Guerinot, 2002). Recently the ferric-chelate reductase-defective (FRD) mutants frd3-1 and frd3-2 were found to be allelic to man1 (which has been renamed frd3-3). The frd3 phenotype is the result of a mutation in FRD3, a member of the multidrug and toxic compound extrusion (MATE) transporter family (Rogers & Guerinot, 2002). It is suggested that FRD3 does not act as a metal transporter directly, but may be involved in regulating whole-plant Fe localization. The Mn hyperaccumulation phenotype is probably caused by the high constitutive expression of IRT1 and possibly the high reductase activity, which could potentially increase the availability of Mn(II). The scope for identifying novel mutants has increased with recent technologies for high-throughput ‘ionomic’ screening, whereby alterations in the nutrient concentrations of a mutant plant are determined by inductively coupled plasma mass spectroscopy (ICP-MS) and ICP atomic emission spectroscopy (ICP-AES) (Lahner et al., 2003). A variety of fast neutron-mutagenized Arabidopsis lines with altered Mn concentrations have already been identified from this approach, including another allele of frd3; however, most of the genes disrupted in these mutations remain to be identified. An increasing number of T-DNA mutagenized knockout lines have also been characterized (information available on the Purdue Ionomics Database http://hort.agriculture.purdue.edu/Ionomics/database.asp; also see Salt, 2004). A number of these knockout lines have significant alterations in Mn concentrations, and implicate some interesting genes in Mn homeostasis. It is also interesting that very few of the fast neutron-mutagenized plants are significantly altered in a single element, including Mn, suggesting coordination in metal homeostasis. For example, many mutants with significantly elevated Mn concentrations are also elevated for cobalt (Co), suggesting interplay between these metals.

In addition to mutant plants, the identification of plants that can hyperaccumulate various metals has begun to increase our understanding of how plants can adapt to excess metal conditions. Of the few hundred metal-hyperaccumulating plants that have been identified, to date 12 of these are Mn hyperaccumulators, with the ability to accumulate and tolerate > 10 000 µg Mn g−1 dry weight, and are mostly woody shrub or tree species growing in subtropical regions (Bidwell et al., 2002; Xue et al., 2004). For some Mn hyperaccumulators, certain biochemical properties have been associated with metal hyperaccumulation. Organic acids can chelate metal ions, and these metal complexes are then thought to be stored in the vacuole. For example, the Mn-hyperaccumulating tree species Austromyrtus bidwillii has an extremely high excess concentration of various organic acids, including oxalic, succinic, malic and malonic acids, which are approximately 3 times the molar equivalent of total Mn in leaf tissue (Bidwell et al., 2002). The characterization of Mn-hyperaccumulator species should ultimately allow the identification of the genes that confer the Mn stress tolerance, although as yet the genetic mechanisms underlying the Mn-hyperaccumulation traits remain to be determined.

Conclusions and future perspectives

  1. Top of page
  2. Summary
  3. Introduction
  4. Mechanisms of Mn2+ accumulation into the cell
  5. Whole-plant Mn2+ uptake characteristics compared with emerging insights from the molecular data
  6. Mechanisms of Mn2+ accumulation into endomembrane compartments
  7. Mechanisms of Mn2+ efflux from the cell
  8. Regulation of Mn2+ transport
  9. Identification of molecular determinants of Mn2+ specificity
  10. Mn homeostasis mutants and hyperaccumulators
  11. Conclusions and future perspectives
  12. Acknowledgements
  13. References

This article has shown that a wide variety of metal transporter family members have the ability to transport Mn2+ in plant cells. This is similar to the situation in S. cerevisiae, where a diverse range of transporters mediate Mn2+ transport (Fig. 1a). However, significant differences exist between yeast and plants as various types of metal transporter, including cation/H+ antiporters and CDF transporters that do not appear to be involved in Mn2+ transport in yeast, can facilitate plant Mn2+ transport (Fig. 1b). It is also apparent that many of these plant proteins are broad-specificity transporters. For instance, IRT1, AtNramp3 and OsYSL2 are Fe-deficiency-regulated Fe2+ transporters that can also transport Mn2+ (Vert et al., 2002; Thomine et al., 2003; Koike et al., 2004), while CAX2 and ECA1 were originally identified as Ca2+ transporters, but have subsequently been shown to also transport Mn2+ (Wu et al., 2002; Pittman et al., 2004b). As described above, this broad selectivity of Mn2+–Ca2+ and Mn2+–Fe2+ transporters can be explained in part by the similar characteristics of these cations. The S. hamata vacuolar transporter ShMTP1 is an intriguing exception as it appears to function specifically as a Mn2+ transporter (Delhaize et al., 2003). It will be particularly interesting to see if ShMTP1 homologues from other plants are also as specific for Mn2+. The substrate specificity of many of these transporters was determined by heterologous expression, often in S. cerevisiae. One must be cautious in the interpretation of such experiments, as the ability of these proteins to transport a particular cation does of course not necessarily mean that this cation transport is physiologically relevant in the plant. Gene deletion or overexpression studies have been instrumental in confirming the involvement of a small number of transporters in Mn homeostasis in the plant (Wu et al., 2002; Delhaize et al., 2003); however, similar studies must be performed for many more of the putative Mn2+ transporters. Furthermore, direct transport studies, such as those using radiolabelled metals (Shigaki et al., 2003; Pittman et al., 2004b), are needed to unequivocally confirm that these proteins do transport Mn2+ or Mn2+-chelate complexes rather than having an indirect role.

Unlike the situation in bacteria and cyanobacteria (Yamaguchi et al., 2002), plant genes involved in Mn sensing have yet to be determined. Similarly, it is unclear whether metallochaperones exist in plants that are specific to Mn2+. Further work is still required to determine the mechanisms for Mn2+ efflux out of the cell, and for Mn2+ accumulation into the Golgi, mitochondria and chloroplast, and the mechanisms by which it is targeted to the particular Mn2+-dependent enzymes in these locations. Further characterization of the currently identified transporters is also required in order to determine the relative contributions of these transporters in Mn homeostasis at both the cellular and whole-plant levels. For example, the cell and tissue expression patterns will vary amongst these Mn2+ transporters. Moreover, the regulatory properties in response to Mn status will differ amongst transporters. Regulation of Mn homeostasis is vital to allow the cell to correctly balance its Mn requirements and to avoid toxicity. It is therefore critical to understand these regulatory mechanisms. Further comparative analysis of the model bacterial and yeast organisms will be important in identifying some of the gaps in plant Mn homeostasis; however, it is quite likely that unique plant-specific genes that cannot be identified by sequence homology alone will also be involved in Mn nutrition. Strategies to identify such novel components could include random mutagenesis screening and functional screening, as well as transcriptomic and proteomic analyses.

In summary, this review has charted the progress that has been made in identifying and characterizing components in Mn homeostasis, and has highlighted some of the future directions that are needed to allow us to fully understand how this important metal is acquired and regulated in plants.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Mechanisms of Mn2+ accumulation into the cell
  5. Whole-plant Mn2+ uptake characteristics compared with emerging insights from the molecular data
  6. Mechanisms of Mn2+ accumulation into endomembrane compartments
  7. Mechanisms of Mn2+ efflux from the cell
  8. Regulation of Mn2+ transport
  9. Identification of molecular determinants of Mn2+ specificity
  10. Mn homeostasis mutants and hyperaccumulators
  11. Conclusions and future perspectives
  12. Acknowledgements
  13. References